The homeobox Nkx2-5, discovered in the host laboratory, encodes a key transcriptional regulator of cardiac development in vertebrates, and mutations in human NKX2-5 are associated with congenital cardiac disease. We will undertake a molecular dissection of one functional domain within Nkx2-5, a transcriptional activation domain within the C- terminus (C-TAD) which is bipartite in structure, requiring both a conserved GIRAW motif and an upstream tyrosine-rich domain. We hypothesize that C-TAD is important for Nkx2-5 activity and regulation in hear development. The proposal seeks to characterize in detail C-TAD activity in vitro and in vivo, and to isolate interacting proteins. We will: 1) perform mutagenesis of individual and multiple tyrosines to assess their importance; 2) assess whether C-TAD tyrosines are phosphorylated using tandem mass spectrometry; 3) assess whether C-TAD influences synergistic interaction act between Nkx2-5 and the cardiac transcription GAT4; 4) assess the significance of point mutations in the GIRAW motif and tyrosines for Nkx2-5 function in ES cell-derived embryoid body cultures and in chimeric mice; and 5) use the yeast 2-hybrid system to isolate interacting proteins.